Raman spectroscopy is a spectroscopic technique which can provide vibration spectrum of molecules. Based upon this, it can detect and get the information of structure and composition of samples at the molecular level because of changes in histiocyte inevitably lead to structural and compositional variation in both intracellular and extracellular biomolecules. Due to its weakness, however, normal Raman signal for detection needs to be enhanced by means of certain techniques, one of which is surface enhanced Raman spectroscopy (SERS). SERS is a technique that enhances Raman scattering by 104-106 times through molecules adsorbed on rough metal (e.g. Au, Ag, Cu and Pt) surfaces. In theory. SERS is highly surface-sensitive enough to detect single molecule. On the other hand, use of near infrared ray excitation (785 nm) in SERS result in detection power lower than 1 mW, realizing nondestructive testing of biological samples.
Membrane electrophoresis is an electrophoretic technique especially suitable for the separation and analysis of body fluid like blood. So far it has wide application in the segregation analysis of serum protein, hemoglobin, globulin, lipoprotein, glucoprotein, alpha fetoprotein, steroids and isozyme. Compared with other electrophoretic techniques such as polyacrylamide gel electrophoresis and agarose gel electrophoresis, membrane electrophoresis has the following advantages:
(1) Fast and time-saving. Take cellulose acetate membrane electrophoresis for instance, less chamber buffer cellulose acetate membrane contains reduces electrodialysis, current thus is mainly conducted by samples, making it fast to separate. Also shorter time will be used for electrophoresis (40-60 min).
(2) High sensitivity and sample-saving. Take blood for instance, only 0.1-2 μl serum is enough for a clear separation band.
(3) High specificity. Take cellulose acetate electrophoresis for instance, there is no tailing because few impurities are adsorbed by cellulose acetate film. Specificity thus can be increased by clear separation band obtained through complete fading of background after staining.
The principle of membrane electrophoresis is explained as follows:
(1) Take separation and analysis of serum protein for example. As is known, the isoelectric point of most serum protein is less than pH of 7.0. In barbital buffer solution with pH of 8.6, serum protein electrified by the anions it ionizes can move to the oppositely charged electrodes under the influence of the electric field, which is known as electrophoresis.(2) In general, membrane electrophoresis is named after the film substrate used. For instance, cellulose acetate electrophoresis is named after the cellulose acetate film substrate. In a condition with the same pH value, charges on different kinds of serum proteins vary resulting from different composition, molecular weight, isoelectric point and shape of the serum protein amino acid. Proteins with less molecular weight carry more charges, and move faster. Whereas proteins with greater molecular weight carry less charges, and move slower. In this way, serum protein can be separated via difference of speeds.
Pathological changes histiocytes undergo often display in three kinds of biomolecules, that is protein, DNA and RNA. But components of body fluid and secretion is complicated. Take blood for example, it comprises of not only serum protein, but also glucose, fat, hormone and xenobiotics such as pharmaceuticals, bacteria and virus. It is difficult to further analyze protein, DNA and RNA using SERS spectrum with traditional approaches which focus on direct SERS detection because SERS spectrum is so sensitive that Raman signals of every part of samples is enhanced, disturbing SERS signals emitted by protein, DNA and RNA in the body fluid.